1. Field of the Invention
The present invention is in the field of genetic engineering and molecular biology. In particular, the invention is directed to transgenic animals and transfected cell lines expressing a protein associated with Alzheimer's Disease, neuroectodermal tumors, malignant astrocytomas, and glioblastomas. This invention is also directed to the use of such transgenic animals and transfected cell lines to screen potential drug candidates for treating or preventing Alzheimer's disease. The invention also relates to new antisense oligonucleotides, ribozymes, triplex forming DNA and external guide sequences that can be used to treat or prevent Alzheimer's disease.
2. Related Art
Alzheimer's disease (AD) (Khachaturian, Z. S., “Diagnosis of Alzheimer's Disease,” Arch. Neurol. 421:1097–1105 (1985)) is the most prevalent neurodegenerative disease and the most common cause of dementia in the Western hemisphere. AD neurodegeneration is characterized by prominent atrophy of corticolimbic structures with neuronal loss, neurofibrillary tangle formation, aberrant proliferation of neurites, senile plaques, and βA4-amyloid deposition in the brain (Khachaturian, Z. S.). Approximately 90 percent of AD occurs sporadically. The cause is unknown, but the most important overall risk factor is aging (Takman, A., “Epidemiology of Alzheimer's Disease. Issues of Etiology and Validity,” Acta Neurol. Scand. Suppl. 145:1–70 (1993)). The apolipoprotein ε4 genotype (Corder, E. H. et al., “Gene Does of Apolipoprotein E Type 4 Allele and the Risk of Alzheimer's Disease in Late Onset Families, Science 261:921–923 (1993)) and a family history of Trisomy 21 Down syndrome (Lai, F. and Williams, R. S., “A Prospective Study of Alzheimer Disease in Down Syndrome,” Arch. Neurol. 46:849–853 (1989) increase risk or accelerate the course of sporadic AD. Familial forms of AD, which account for 5 to 10 percent of the cases, have been linked to mutations in the amyloid precursor protein (APP) gene (Kennedy, A. M. et al., “Familial Alzheimer's Disease. A Pedigree With a Mis-Sense Mutation in the Amyloid Precursor Protein Gene (Amyloid Precursor Protein 717 Valine→ Glycine,” Brain 309–324 (1993); Peacock, M. L. et al., “Novel Amyloid Precursor Protein Gene Mutation (Codon 665Asp) in a Patient with Late-Onset Alzheimer' s Disease,” Ann. Neurol. 35:432–438 (1994); Tanzi, R. E. et al., “Assessment of Amyloid Beta-Protein Precursor Gene Mutations in a Large Set of Familial and Sporadic Alzheimer's Disease Cases,” Am. J. Hum. Genet. 51:273–282 (1992)) located on Chromosome 21 (Robakis, N. K. et al., Chromosome 21q21 Sublocalization of Gene Encoding Beta-Amyloid Peptide in Cerebral Vessels and Neuritic (Senile) Plaques of People with Alzheimer Disease and Down Syndrome,” Lancet 1:384–385 (1987)), or presenilin genes located on Chromosomes 1 and 14 (Levy-Lahad, E. et al., “Candidate Gene for the Chromosome 1 Familial Alzheimer's Disease Locus,” Science 18:973–977 (1995); Sorbi, S. et al., “Missense Mutation of S182 Gene in Italian Families With Early Onset Alzheimer's Disease,” Lancet 346:439–440 (1995); Sherrington, R. et al. “Cloning of a Gene Bearing Missense Mutations in Early-Onset Familial Alzheimer's Disease, Nature 375:754–760 (1995); Rogaev, E. I. et al., “Familial Alzheimer's Disease in Kindreds With Missense Mutations in a Gene on Chromosome 1 Related to the Alzheimer's Disease Type 3 Gene,” Nature 376:775–778 (1995); Barinaga, M. et al., “Candidate Gene for the Chromosome 1 Familial Alzheimer's Disease Locus,” Science 269:973–977 (1995)). Over-expression and abnormal cleavage of APP may promote AD neurodegeneration since all individuals with Trisomy 21 Down syndrome who survive beyond the fourth decade develop AD with extensive central nervous system (CNS) accumulation of βA4-amyloid (Lai, F. and Williams R. S., “A Prospective Study of Alzheimer Disease in Down Syndrome,” Arch. Neurol. 46:849–853 (1989)), and experimentally, βA4-amyloid is neurotoxic and apotogenic (LaFerla, F. M. et al., “The Alzheimer's A Beta Peptide Induces Neurodegeneration and Apoptotic Cell Death in Transgenic Mice,” Nat. Genet. 9:21–30 (1995). In addition, missense mutations in persenilin 1, as occurs in nature, cause vasculopathy and massive accumulations of peptides in the brain (Lemere, C. A. et al., “The E280A Presenilin 1 Alzheimer Mutation Produces Increased Aβ42 Deposition and Severe Cerebellar Pathology,” Nature Med. 2:1146–1150 (1996); Mann, D. M. et al., “Amyloid Beta Protein (Abeta) Deposition in Chromosome 14-Linked Alzheimer's Disease: Predominance of Abeta42 (43),” Ann Neurol. 40:149–156 (1996)).
Central nervous system biochemical and molecular abnormalities identified in AD include: 1) increased phosphorylation of tau and other cytoskeletal proteins in neurons (Grundke-Iqbal, I. et al., “Abnormal Phosphorylation of the Microtubule-Associated Protein τ (tau) in Alzheimer Cytoskeletal Pathology,” Proc. Natl. Acad Sci. U.S.A. 83:4913–4917 (1986)); 2) aberrant expression of genes modulated with neuritic sprouting such as the growth associated protein, GAP-43 (de la Monte, S. M. et al., “Aberrant GAP-43 Gene Expression in Alzheimer's Disease,” Am. J. Pathol. 147:934–946 (1995)), constitutive endothelial nitric oxide synthase (de la Monte, S. M. and Bloch, K. D. “Aberrant Expression of the Constitutive Endothelial Nitric Oxide Synthase Gene in Alzheimer's Disease,” Molecular and Chemical Neuropathy 29: (in press)) transforming growth factor β (Peress, N. S. and Perillo, E., “Differential Expression of TGF-beta 1, 2, and 3 Isotypes in Alzheimer's Disease: a Comparative Immunohistochemical Study With Cerebral Infarction, Aged Human and Mouse Control Brains,” J. Neuropathol. Exp. Neurol. 54: 802–811 (1995)), and metallothionine-3 (Aschner, M. “The Functional Significance of Brain Metallothioneins,” Faseb. J. 10:1129–1136 (1996)); 3) increased expression of genes associated with glial cell activation, such as glial fibrillary acidic protein (Goodison, K. L. et al., “Neuronal and Glial Gene Expression in Neocortex of Down's Syndrome,” J. Neuropathol Exp. Neurol. 52:192–198 (1993)) and alpha-1 antichymotrypsin (Pasternack, J. M. et al., “Astrocytes in Alzheimer's Disease Gray Matter Express Alpha 1-Antichymotrypsin mRNA,” Am. J. Path. 135:827–834 (1989); and 4) altered expression of genes that protect neurons from either cytotoxic or programmed cell death, including sulfated glycoprotein-2 (May, P. C. et al., “Dynamics of Gene Expression for a Hippocampal Glycoprotein Elevated in Alzheimer's Disease and in Response to Experimental Lesions in Rat,” Neuron 5:831–839 (1990), cathepsin D (Cataldo, A. M. et al., “Gene Expression and Cellular Content of Cathepsin D in Alzheimer's Disease Brain: Evidence for Early Up-Regulation of the Endosomal-Lysosomal System,” Neuron 14:671–680 (1995)), superoxide dismutase 1 (Somerville, M. J. et al., “Localization and Quantitation of 68 kDA Neurofilament and Superoxide Dismutase-1 mRNA in Alzheimer Brains, Brain Res. Mol. Brain Res. 9:1–8 (1991), mitochondrial cytochrome oxidase (Chandrasekaran, K. et al., “Impairment in Mitochondrial Cytochrome Oxidase Gene Expression In Alzheimer Disease,” Brain Res. Mol. Brain. Res. 24:336–340 (1994)), C1q component of complement (Fischer, B. et al., “Complement C1q and C3 mRNA Expression in the Frontal Cortex of Alzheimer's Patients,” J. Mol. Med. 73:465–471 (1995)), Calbindin D28k (Yamagishi, M. et al., “Ontogenetic Expression of Spot 35 Protein (Calbindin-D28k) in Human Olfactory Receptor Neurons and its Decrease in Alzheimer's Disease Patients,” Ann. Ontol. Rhinol. Laryngol. 105:132–139 (1996), and bcl-2 (O'Barr, S. et al, “Expression of the Protooncogene bcl-2 in Alzheimer's Disease Brain,” Neurobiol. Aging 17:131–136 (1996).
In previous studies, we demonstrated increased immunoreactivity in AD brains using a polyclonal antisera prepared against a pancreatic protein (Ozturk, M. et al., “Elevated Levels of an Exocrine Pancreatic Secretory Protein in Alzheimer's Disease Brain,” Proc. Natl. Acad. Sci. U.S.A. 86:419–423 (1989); de la Monte, S. M. et al., “Enhanced Expression of an Exocrine Pancreatic Protein in Alzheimer's Disease and the Developing Human Brain,” J. Clin. Invest 86:1004–1013 (1990); WO90/06993). Using such polyclonal antibodies, we isolated the AD7c-NTP cDNA from an AD brain expression library (WO94/23756). In WO94/23756, this clone is also referred to as AD10-7, which was deposited in DH1 cells at the ATCC under accession no. 69262. The nucleotide sequence of this cDNA is shown in FIG. 16R of WO94/23756. However, this sequence comprises numerous errors. See also WO96/15272 (Seq. ID No. 120, pages 168–170), which also comprises numerous errors. As a result, the predicted amino acid sequence (Seq. ID No. 121; WO96/15272) is also wrong.